Phage with nuclear localization signal

ABSTRACT

A λ phage with a nuclear localization signal has been obtained by constructing a vector capable of expressing a fused protein between a gpD protein constituting the head of a λ phage and a nuclear localization signal sequence, transforming  Escherichia coli  with this vector, and propagating a mutant λ phage which cannot express the gpD protein in  E. coli  in this transformant. It has been confirmed that the resulting λ phage is capable of packaging λ phage DNAs of 80% and 100% genome sizes. After further confirming that the nuclear localization signal exposed on the outside of the head of this phage, this phage has been microinjected into cells to analyze its nuclear localization activity. Thus, it has been clarified that this phage has a nuclear localization activity.

The present application is a Division of and claims priority to U.S.Ser. No. 09/615,283, filed Jul. 13, 2000, now U.S. Pat. No. 6,300,120,which claims priority to and is a Division of U.S. Ser. No. 09/242,131,filed Sep. 10, 1999, Now U.S. Pat. No. 6,235,521, which was a NationalStage of International Application PCT/JP96/03861, filed Dec. 27, 1996and benefit of Japanese patent application 8/227787, filed on Aug. 9,1996, the disclosures of which are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to the field of genetic engineering,especially to the transportation of exogenous materials by means ofvirus particles.

BACKGROUND ART

The gene transfer technology to artificially introduce an exogenous geneinto cells is an important technology not only as a fundamentaltechnology to analyze a variety of biological phenomena but also as onewhich leads to useful applications such as gene therapy and productionof beneficial animals. Generally, two methods have been used for genetransfer. One is a biological method using a virus having an exogenousgene, and the other is a physical method in which an exogenous gene isphysically introduced into cells.

The method using a virus is based on the principle that a cell isinfected with a recombinant virus in which the gene of interest isincorporated, and the entire recombinant virus genome integrates intothe genome of the host cell. This method is currently attracting muchattention as a technological basis for gene therapy for such diseases asLesch-Nyhan syndrome and adenosine deaminase (ADA) deficiency. However,it has been pointed out that the method has various problems such as thepathogenicity of the virus since it utilizes the biological propertiesof the virus itself. For this reason, modified retroviral vectorswithout the regions associated with the viral pathogenicity andreplication have currently being developed. However, these modifiedvectors have yet many problems that they might still cause someundesirable effects on cells, and they can infect only dividing cells.

Therefore, physical methods to introduce non-viral vectors are now usedas well as the above-mentioned methods using viruses. In one of theestablished physical methods, non-viral vectors are introduced intocells in combination with chemicals such as calcium phosphate,DEAE-dextran, polycations, or liposomes. However, these physical methodshave such problems that the transfection efficiency of genes into cellsis low, and that the exogenous gene on a non-viral vector thustransfected does not reach the cell nucleus in many cases. Therefore,the methods have many difficulties to be overcome for being applied togene therapy.

Recently, it was reported that the proteins which are transported intothe nucleus of eukaryotic cells and function there have a specific aminoacid sequence that functions as a signal (NLS: nuclear localizationsignal) for their transportation into the nucleus (G. Garcia-Bustos etal., Biochem. Biophys. Acta 1071: 83-101 (1991)). Moreover, it was alsoreported that attaching the nuclear localization signal to a proteinthat normally does not translocate to the nucleus will confer thenuclear translocation activity on this protein (R. E. Lanford et al.,Cell 46: 575-582 (1986), Y. Yoneda et al., Exp. Cell. Res. 170: 439-452(1987), D. Chelsky et al., Mol. Cell. Biol. 9: 2487-2492 (1989)). Basedon this knowledge, researches have been made using the nuclearlocalization signal so that the gene introduced by physical methods canreach the nucleus with a high probability. That is, the techniques arestudied to condense DNA as close as possible to 40 nm, the size of thenuclear membrane pore, attach the nuclear localization signal to thiscondensate, and thereby actively transport the DNA to the nucleus. Forexample, efforts have been made to make DNA more compact by usingproteins such as HMG-1 and histones, as well as poly-L-lysines (Jose C.Perales et al., E. J. B. 266: 255-266 (1994)), and cationic liposomes(J. Zabner et al., J. B. C. 270: 18997-19007 (1995)).

However, the synthetic chemical approach had problems with solubilityand homogeneity of the complex with DNA, and with the varying degrees ofcondensation of DNA dependent on the salt concentration. Moreover,construction of the complex is possible only under highly alkalineconditions and impossible under physiological conditions, which has beenone of the problems to be solved for practical use.

It has been suggested that, on the viruses that infect animals such asadenovirus and SV40, the nuclear localization signals exist in theircapsid proteins, and they function to actively translocate their DNA atthe early stage of infection (Urs. F. Greber and Harumi Kasamatsu,Trends in Cell Biology 6: 189-195 (1996)). It has been also suggestedthat the SV40 particle with its diameter of 45 nm invade the nucleus inthe form of virion (K. Hummeler et al., J. Virol. 6: 87-93 (1970)).Furthermore, MS-2 phage is reported to have a transport system in whichexogenous substances are enveloped by the capsid (InternationalApplication published in Japan No. Hei-508168). However, any transportsystem using virus particles, which is capable of using long chain DNAand translocating the DNA into the nucleus, has not been reported.

DISCLOSURE OF THE INVENTION

An objective of the present invention is to provide a system thatenables delivering genes introduced into cells to the nucleus. Morespecifically, the objective of the invention is to provide a λ phagewith a nuclear localization signal exposed on the outer surface of itshead, and capable of packaging long chain DNA.

In order to translocate long chain DNA into the nucleus, it is necessaryto condense the DNA to nearly 40 nm, the size of nuclear membrane pore.The present inventors paid attention to the head of a λ phage, which isable to compactly package desired long chain DNA in vitro and to protectthe DNA from the attack by external DNases, and used it as a carrier ofthe DNA. Furthermore, we paid attention to the phenomena that theviruses that infect animals can invade the nucleus in the form of virionin virtue of nuclear localization signals in their capsid proteins, andattempted to actively transport DNA into the nucleus by preparing andusing the λ phage head to which a nuclear localization signal has beenattached. More specifically, we used the following steps.

First, we constructed a vector that expresses a fusion protein betweenthe gpD protein, which is one of the proteins to constitute the λ phagehead, and the nuclear localization signal sequence, transformedEscherichia coli with this vector, then infected the transformants witha mutant λ phage incapable of expressing gpD in the E. coli cells(hereinafter designated as “D amber phage”). By plaque formationanalysis and western blot analysis using an anti-gpD antibody, we haveconfirmed that the mutant phage was complemented by the fusion proteinbetween the gpD protein and the nuclear localization signal sequenceexpressed by the vector and, a λ phage having the nuclear localizationsignal attached to its head was obtained. That is, we have found thatthe fusion protein expressed in E. coli has been complementarilyintegrated into the phage head which does not express the protein.

Next, we have obtained a similar result by introducing the vector thatexpresses the fusion protein between the gpD protein and the nuclearlocalization signal sequence into the E. coli lysogenized by the mutantλ phage, and by heat-inducing the lysogenic phage. More specifically, weintroduced the vector that expresses the above fusion protein into theE. coli lysogenized by the D amber phage, and heat-induced thetransformants. As the result, the phage whose head has not incorporatedthe fusion protein and consists of the gpE protein became sensitive toEDTA, while the phage which has incorporated the fusion proteinexhibited resistance to EDTA. Next, we treated the resulting phage withEDTA and measured the titer. As a result, it was revealed that the phagepackaged with 80% genome size DNA was constructed, and that the fusionprotein was incorporated in the phage head. In addition, we haveconfirmed that the phage had the nuclear localization signal exposed onthe outer surface of the head. We also confirmed that the phageincorporating the fusion protein was formed in the same manner even whenwe used 100% genome size DNA. Furthermore, we introduced the phagehaving the nuclear localization signal exposed on the outer surface ofits head into HEL-R66 cells, which are human fetal lung cells, bymicroinjection and proved that the phage has a nuclear translocationactivity, thereby completing the present invention.

Therefore, the present invention relates to a λ phage capable ofpackaging macromolecules such as long chain DNA and having a nucleartranslocation activity.

More specifically, it relates to:

(1) a phage or its head having a protein containing a nuclearlocalization signal as a component of the head,

(2) the phage or its head of (1), wherein said nuclear localizationsignal comprises any one of the sequences described in SEQ ID NO: 1 toSEQ ID NO: 4.

(3) the phage or its head of (1), wherein said phage is a λ phage,

(4) the phage or its head of (3), wherein said protein containing thenuclear localization signal is a fusion protein between the nuclearlocalization signal and a phage head protein,

(5) the phage or its head of (4), wherein said phage head protein is Dprotein of a λ phage,

(6) a fusion protein between a nuclear localization signal and a proteinthat forms a phage head,

(7) the fusion protein of (6), wherein said nuclear localization signalcomprises any one of the sequences described in SEQ ID NO: 1 to SEQ IDNO: 4.

(8) the fusion protein of (6), wherein said phage is a λ phage,

(9) the fusion protein of (6), wherein said phage head protein is the Dprotein of a λ phage,

(10) a DNA encoding any one of the proteins of (6) to (9),

(11) a vector containing the DNA of (10),

(12) a bacterial host carrying the vector of (11), (13) the bacterialhost of (12), wherein said host is Escherichia coli,

(14) a kit for transforming cells, wherein said kit comprises thebacterial host of (12) or (13), and (b) a phage from which a headprotein contained in a fusion protein expressed in said host has beenderived, wherein said phage cannot express said head protein in saidbacterial host,

(15) the kit of (14), wherein said phage is a λ phage,

(16) the kit of (14), wherein said head protein contained in the fusionprotein expressed in the bacterial host is D protein of a λ phage,

(17) a method for translocating a desired substance into the nucleus ofa desired cell, wherein said method comprises: (a) packaging into thephage or into its head of (1) the desired substance to be translocatedto the nucleus, and (b) introducing said phage or its head into thedesired cell,

(18) the method of (17), wherein said desired substance is a nucleicacid,

(19) the method of (17), wherein said phage is a λ phage,

(20) the method of (17), wherein said cell is a mammalian cell.

The present invention relates to the technology to package an exogenousmaterial into the head of a phage to which a nuclear localization signalis attached, introduce the phage into a desired cell in which theexogenous material is to function, and translocate the exogenousmaterial together with the phage particle into the nucleus of the targetcell.

The nuclear localization signal used in the present invention is notparticularly limited as far as it has the activity to translocate asubstance to which the signal sequence is attached into the nucleus. Forexample, in the case of translocating the λ phage particle into thenucleus, it is preferable to use the nuclear localization signal of SV40VP1, SV40 large T antigen, or hepatitis D virus δ antigen, or a sequencecontaining “PKKKRKV” (SEQ ID NO: 4)(by the single letter representationof amino acids as is found in Encyclopedia of Biochemistry, 2nd ed.)that is the minimum unit having the nuclear translocation activitywithin the nuclear localization signal of SV40 large T antigen.

The phage used in the present invention is not particularly limited asfar as an exogenous material can be packaged into its head. Phages suchas a λ phage and an M13 phage can be used.

A number of methods can be used to prepare the phage whose head isconstituted by a protein containing the nuclear localization signal. Forexample, one can chemically bind the nuclear localization signalsequence to a phage head protein, or combine a DNA encoding the nuclearlocalization signal sequence with gene encoding a phage head protein andincorporate it into a vector, express it as a fusion protein a bacterialhost, and proliferate in the host a mutant phage that cannot express thehead protein, thereby constructing the phage head. There are nolimitation to the vectors that can be used in the above methods, andvarious vectors can be used. The bacterial host is not particularlylimited as far as the phage used in the method can proliferate in thehost. For example, when a λ phage is used, a variety of E. coli strainsin which the phage can proliferate can be used. The nuclear localizationsignal sequence may chemically bind to the phage head protein, directlyor via a cross-linking agent or a spacer peptide. The DNA encoding thenuclear localization signal sequence and the gene encoding a phage headprotein may be combined directly or through a spacer nucleotide.

The head protein used in the above methods non-limitedly include gpDprotein or gpE protein when the phage is a λ phage, and gene 3 proteinwhen the phage is M13.

In the present invention, the phage is introduced into the cell afterpackaging an exogenous material. As for packaging, the method of Ishiuraet al. (Gene 82: 281-289 (1989)) or the method of Sternberg et al.(Japanese Patent No. Hei 59-500042) can be used. As for the exogenousmaterial, a gene, a gene fragment, ribozyme, an antisense gene, or anyother substance to make it function in the nucleus can be used. Forexample, when gene therapy is performed, it will be effective to use thenormal counterpart of a defective gene. When the function of a specificgene is analyzed, it will be effective to use an antisense gene againstthe gene. Also, if one wishes to create transgenic animals, it will beeffective to introduce a gene which is associated with the phenotype tobe conferred into them. It should be noted that the present inventionenables packaging a long chain nucleic acid such as a gene with itsupstream region.

The method to introduce the phage that has packaged an exogenousmaterial, includes the microinjection method, the lipofection method,the liposome method, the HVJ-liposome method, the immuno-liposomemethod, the pH-sensitive liposome method, the erythrocyte ghost method,the DEAE-dextran method, the method utilizing endocytosis of a receptoron the cell surface, the method utilizing a specific antigen on the cellsurface, the method utilizing a synthetic macromolecular carrier, themethod utilizing a particle gun, etc. There is no particular limitationto the cells into which the phage packaging an exogenous material isintroduced, and various cells can be used depending on the purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of fusion proteins between various nuclearlocalization signals (SEQ ID NO: 7-12) and the gpD protein, which is a λphage head protein.

FIG. 2 (Panels 1-4) shows microscopic photographs indicating the nucleartranslocation activity of the λ phage to which a nuclear localizationsignal was attached via a cross-linking agent.

FIG. 3 shows microscopic photographs indicating the nucleartranslocation activity of the λ phage having a nuclear localizationsignal exposed on the surface of its head.

BEST MODE FOR IMPLEMENTING THE INVENTION

The present invention is described in more detail with reference to thefollowing examples, but is not construed to be limited thereto.

Example 1 Construction of a λ Phage With a Nuclear Localization Signal

Using a wild type λ phage gene as a template, cDNA for the gpD protein,which is one of the proteins to constitute the λ phage head, was clonedby PCR. The PCR was done according to the method of Sternberg et al.(Sternberg et al., PNAS 92: 1609-1613 (1995)). More specifically,5′-GTAAGCCATGGTTATGACGAGCAAAG-3′(which contains an NcoI site at the 6thto 11th nucleotide residues from the 5′side) (SEQ ID NO: 5) and5′-GTTCGAATTCCTATTAAACGATGCTGATTGCC-3′(which contains an EcoRI site atthe 5th to 10th nucleotide residues from the 5′side) (SEQ ID NO: 6) wereused as primers, and a fragment of about 4 kb containing the gpD gene,which was generated by digesting 20 μg of the λ phage genome (TOYOBO,7.9 OD/ml) with ApaI and ApaLI, was used as a template. The PCR reactionwas done using 0.2 μg of the template, 10× Reaction buffer (Pharmacia;500 mM KCl, 15 mM MgCl₂, 100 mM Tris-HCl (pH 9.0)), 1 μM each ofprimers, 50 μM dNTP, 100 μl of 5U Taq DNA polymerase (Pharmacia) byperforming 25 cycles of the steps containing heat denaturation at 90° C.for 3 min, annealing at 55° C. for 2 min, and extension at 72° C. for 3min and finally one cycle of heat denaturation at 90° C. for 3 min,annealing at 55° C. for 2 min, and extension at 72° C. for 10 min. TheDNA fragment amplified by the above PCR was introduced into the NcoI andEcoRI sites of an E. coli expression vector, pTrcHisA (Invitrogen). Theresulting vector was designated “pTrcHisA-gpD.” After confirming the DNAsequence by the cycle sequencing method, the vector was introduced intoE. coli TOP10 (Seth G. N. Grant et al.,PNAS87: 4645-4649 (1990)), andthe gpD protein was expressed at a high level in the E. coli. Theexpression of the protein was examined by SDS polyacrylamide gelelectrophoresis (SDS-PAGE). As a result, a strong band was detected atthe 11.6 kDa position, which is themolecular weight of the protein, 6hrs after the induction by 1 mM IPTG.

Next, E. coli TOP10 (pTrcHisA-gpD) and E. coli 594 (pTrcHisA-gpD), whichare expressing the gpD protein because “pTrcHisA-gpD” has beenintroduced and which does not contain a suppressor mutation (sup⁰), wereinfected with the D amber phage, and the plaque forming activity and thetiter were examined. The results indicated that plaques were formed inboth cases and that the titers were equivalent to the one with LE392,which contains a suppressor mutation against D amber. Therefore, it wasdemonstrated that the phage is formed by functional complementation evenwhen the gpD gene exists in trans.

Then, the phage with a nuclear localization signal was constructed byexpressing a fusion protein between a nuclear localization signal andgpD in E. coli. As the nuclear localization signal, in addition to thethree types of nuclear localization signal from SV40VP1, SV40 large Tantigen, and hepatitis D virus δ antigen (SEQ ID NOS: 1, 2, and 3respectively), all of which were confirmed to be effective by thepresent inventors, two other types of nuclear localization signal, whichwere (1) a fusion protein between the minimum unit of the nuclearlocalization signal “PKKKRKV (SEQ ID NO: 4)” and a spacer protein, and(2) the minimum unit of the nuclear localization signal “PKKKRKV” (SEQID NO: 4) alone, were used. Also, a fusion protein between an 8polypeptide from angiotensin II (which is not a nuclear localizationsignal) described in Sternberg et al., PNAS 92: 1609-1613 (1995) and aspacer protein was used in order to confirm the phage formationcapability, making the total of six types (FIG. 1). Next, theoligonucleotides corresponding to these nuclear localization signalswere synthesized, and introduced into the NcoI site of theabove-described “pTrcHisA-gpD”. After confirming that the plasmids werecorrectly constructed by cycle sequencing method, the vectors wereintroduced into E. coli Top10, and the fusion proteins between thenuclear localization signals and the gpD protein were expressed at ahigh level in the E. coli. The expression of the fusion proteins wasexamined by SDS polyacrylamide gel electrophoresis (SDS-PAGE). As aresult, a strong band was detected at the expected position for themolecular weight of the desired protein 6 hrs after the induction by 1mM IPTG. Further, the plaque formation capability was examined byinfecting the bacterial cells with the D amber phage, and plaqueformation was observed when three types of peptides, SV40 large Tantigen, hepatitis D virus δ antigen, and a fusion protein between the 8polypeptide from angiotensin II and a spacer protein, were used. Thetiter measurements indicated that all the phage had a titer on the orderof 10¹⁰, but that the plaque forming time was 10 hrs for SV40 large Tantigen, and 18 hrs for hepatitis D virus δ antigen, showing delayscompared with the 6 hrs which is normal plaque forming time (Table 1).

TABLE 1 Plaque forming Host E. coil strain Titer (PFU/ml) time LE392(supE44, supF58) 1.8 × 10¹⁰ 6 hrs TOP10 (sup°)/ 0.5 × 10¹⁰ 6 hrspTrc-gpD (no induction) TOP10 (sup°)/ 0.6 × 10¹⁰ 6 hrs pTrc-angiotensinII-gpD (IPTG induction) TOP10 (sup°)/ 0.4 × 10¹⁰ 10 hrs  pTrc-SV40 largeT antigen-gpD (IPTG induction) TOP10 (sup°)/ 0.2 × 10¹⁰ 18 hrs pTrc-hepatitis D virus δ antigen-gpD (IPTG induction)

It was also confirmed that the phage particle thus obtained contain thefusion proteins between the nuclear localization signals and gpD by thewestern blotting method.

Example 2 Packaging of the λ Phage Genome by the λ Phage With a NuclearLocalization Signal

In place of the above method in which the phage was formed by theinfection of E. coli TOP10, the method expected to provide better phageformation capabilities in which a lysogenic phage (E. coli 594) or an80% genome phage is heat-induced were used in the following. E. coli 594(λkDam15 cIts857 Sam7) is lysogenized by a 100% genome phage, and has atemperature sensitive repressor cI which is inactivated by the treatmentat 42° C. for 15 min. Because of the D amber mutation, however, the headcannot be produced and only the tail is produced in this sup⁰ strain ofE. coli. “pTrcHisA-gpD” was introduced into this E. coli strain to allowthe expression of the fusion proteins, and the phage formation wasexamined by heat induction. As a result, the phage was formed when anyone of the six types of peptides as described above was used. Theresults indicated that the fusion proteins were incorporated into thehead of the phage in this E. coli strain lysogenized by a 100% genomephage. The titers of the resulting phage are shown in Table 2.

TABLE 2 Titer (PFU/ml) (analyzed with Proteins expressed E. coil LE392)— 0 gpD   5 × 10⁸ SV40 large T antigen-gpD 3.6 × 10⁸ fusion proteinSV40VP1-gpD fusion protein 3.0 × 10⁸ hepatitis D virus δ antigen-gpD 3.3× 10⁸ fusion protein angiotensin II-spacer-gpD   5 × 10⁸ fusion proteinPKKKRKV-spacer-gpD fusion protein 1.7 × 10⁸ (SEQ ID NO:11) PKKKRKV-gpDfusion protein 8.9 × 10⁷ (SEQ ID NO:12)

Next, another set of experiments were performed using E. coli 594lysogenized by an 80% genome D amber phage. cI, which is the repressorof the phage and encoded by the phage, is temperature sensitive, andbecomes inactive by a treatment at 42° C. for 15 min, which results inlysis of the bacteria by the phage. In addition, since the phagecontains the D amber mutation, it cannot express gpD in this sup⁰ host,and the head is usually formed only with gpE. (There are two kinds of λphage head proteins-gpD and gpE.) The phage having only gpE is extremelysensitive to EDTA. On the other hand, if the E. coli is allowed toexpress the fusion protein and the heat-induced phage incorporates thefusion protein, it would show EDTA resistance. “pTrcHisA-gpD” wasintroduced into the E. coli strain, then the phage was prepared on the 8ml scale, and treated with 10 mM EDTA. E. coli LE392 was infected withthem to measure their titers. As a result, the EDTA resistance wasconfirmed using any one of the six types of peptides used in Example 1.The results indicated that the 80% genome phage have higher titers thanthe 100% genome phage, suggesting that the head structure is stabilizedin the former (Table 3).

TABLE 3 Titer (PFU/ml) (analyzed with E. coil LE392) Proteins expressedEDTA (−) EDTA(+) — 4.0 × 10⁹ 0 gpD 8.6 × 10⁸ 6.8 × 10⁸ SV40 large Tantigen-gpD 9.8 × 10⁹ 8.0 × 10⁹ fusion protein SV40VP1-gpD fusionprotein 2.3 × 10⁸ 2.4 × 10⁸ hepatitis D virus δ antigen-gpD 5.6 × 10⁸5.6 × 10⁸ fusion protein angiotensin II-spacer-gpD 7.1 × 10⁸ 8.4 × 10⁸fusion protein PKKKRKV-spacer-gpD fusion protein 2.3 × 10⁸ 2.3 × 10⁸(SEQ ID NO:11) PKKKRKV-gpD fusion protein 1.8 × 10⁸ 1.5 × 10⁸ (SEQ IDNO:12)

Example 3 Nuclear Translocation Activities of the λ Phase to Which theNuclear Localization Signal is Attached Through a Cross-Linking Agent

(1) Preparation of phage particles from λ phage lysogenic bacteria

E. coli w3350thy⁻ (λcI847 Sam7) lysogenized by λ phage was cultured inthe LB (thy) medium at 32° C., and at the logarithmic growth phase(2×10⁸ cells/ml) it was shaken at 45° C. for 25 min to induce the phageproduction. Then, the culture was shaken at 39° C. for 3 hrs,centrifuged at 5,000 rpm for 10 min, and the precipitated E. coli wasresuspended in the SM buffer (0.1 MNaCl, 8mMMgSO₄, 0.01% gelatin, 50 mMTris-HCl (pH 7.5)). The concentrated bacteria were lysed by adding 37°C. chloroform and stirring. Further, the solution was added with DNase,centrifuged at 8,000 rpm for 30 min to remove insoluble matters, and thesupernatant was centrifuged at 23,000 rpm for 60 min to precipitate thephage, and the phage were resuspended in the SM buffer. The recoveredphage particles were purified by cesium chloride density-gradientcentrifugation.

(2) Cross-linking the nuclear localization signal to the λ phage

A cross-linking agent (SMPB; Pierce) was used to cross-link the nuclearlocalization signal to the λ phage. SV40 large T antigen (SEQ ID NO: 2)was used as the nuclear localization signal. The λ phage was prepared at1.1 mg/ml in a buffer (0.1 M NaCl, 8 mM MgSO₄, 20 mM Hepes · NaOH (pH7.0)). 10 mM SMPB dissolved in anhydrous DMSO was added at a 5,000 timesmolar ratio to the phage particles, and the mixture was incubated at 25°C. for 1 hr. The mixture was then dialyzed against a buffer (0.1 M NaCl,8 mM MgSO₄, 20 mM Tris-HCl (pH 7.5)) overnight and the SMPB-modified λphage was obtained by removing the unreacted SMPB. The synthetic nuclearlocalization signal peptide (Sawady Technology) was dissolved atequimolar with SMPB in 20 mM Tris-HCl (pH 8.0) containing 0.1 M NaCl,and DTT was added to 50 mM to the solution, and reduction reaction wascarried out at 37° C. for 1 hr. After removing DTT by gel filtration ina buffer (0.1 M NaCl, 8 mM MgSO₄, 20 mM Hepes · NaOH (pH 7.0)), theeluate was added to the SMPB-modified phage, and allowed to react at 25°C. for 3 hrs. The reaction solution was put into a centrifuge tube so asto layer over a layer of 1 ml of 10% sucrose solution (containing 0.1 MNaCl, 8 MM MgSO₄, 20 mM Hepes NaOH (pH 7.0)) previously placed in thetube, centrifuged at 4° C. at 20,000 rpm (Beckman TLS-55 rotor) for 1hr. The precipitate was resuspended in a buffer (0.1 M NaCl, 8 mM MgSO₄,20 mM Hepes NaOH (pH 7.0)) to recover the phage to which the nuclearlocalization signal was attached.

(3) Detection of the nuclear translocation activity by microinjectionand the indirect fluorescent antibody method

The λ phage with the nuclear localization signal was microinjected intothe cytoplasm of the cells cultured on a cover slip by the methoddescribed in the literature (Y. Yoneda et al., Exp. Cell. Res. 170:439-452 (1987), T. Tachibana et al., J. Biol. Chem. 269: 24542-24545(1994)). After the cells were incubated at 37° C. for 5 min to 4 hrs,PBS (−) containing 3.7% formaldehyde was added to fix the cells at roomtemperature for 20 min. The range of incubation time “15 min to 4 hrs”before the fixation was set in order to measure the time necessary forthe λ phage with the nuclear localization signal to reach the nucleusafter the injection. The cells were treated with 0.5% Triton X-100 for5min at room temperature after the fixation, and immersed in 10% BlockAce (Dainippon Pharmaceutical) for 1 hr at room temperature forblocking. Next, the cells were reacted at room temperature for 1 hr witha 500-fold dilution of the rabbit anti-λ phage serum (obtained from Dr.Hideyuki Ogawa, Osaka University) as the primary antibody, then reactedat room temperature for 1 hr with the FITC-labeled anti-rabbit IgG (6μg/ml) as the secondary antibody, the localization of the λ phage withthe nuclear localization signal in the cell was examined using afluorescent microscope. The results confirmed that the λ phage with thenuclear localization signal translocates to the nucleus immediatelyafter the microinjection (FIG. 2, upper left), and remains in theperiphery of the nucleus at 30 min after microinjection (FIG. 2, upperright). The unmodified λ phage used as a control in this experiment,which does not have a nuclear localization signal, dispersed in thecytoplasm immediately after the microinjection (FIG. 2, lower left), anddiffused almost homogeneously within 30 min (FIG. 2, lower right).

Example 4 Analysis of Surface Exposure of the Nuclear LocalizationSignal on the Phage Head

A cysteine residue at the N-terminal side of the synthetic SV40 large Tantigen was cross-linked with SulfoLink Gel (Pierce) to prepare animmobilized column (2 ml, 5 cm). Ten ml of anti-SV40LT rabbit serum wastreated with saturated ammonium sulfate, and equilibrated by dialysisover the coupling buffer (50 mM Tris-HCl (pH 8.5), 5 mM EDTA-Na). Thiswas applied onto the column, and the binding antibody was eluted in 0.1M Gly-HCl (pH 2.5) fractionated in 0.5 ml portions, and the elutedfraction was neutralized by 0.5 ml of 2 M Tris-HCl (pH 8.0). Ten mg ofthe affinity-purified antibody thus obtained was reacted with 3×10⁸molecules of the 80% genome phage expressing SV40 large T antigen(hereinafter designated as “LT-phage”) at 4° C. overnight. One hundredml of Protein A-Sepharose 4B (Pharmacia, 50% slurry) was added thereto,allowed to react at room temperature for 1 hr for absorption,centrifuged at 5,000 rpm for 5 min, and the titer of the non-absorbedphage in the supernatant was measured using E. coli LE392. As a control,the phage expressing only the gpD protein was similarly analyzed. Inaddition, similar analyses were made using the rabbit y globulin inplace of the anti-SV40LT rabbit serum, and also without using theseantibodies (Table 4)

TABLE 4 Titer (PFU/ml) (analyzed with E. coli LE392) Anti-SV40 large Tantigen Nuclear localization signal Protein antibody Rabbit γ globulinNo antibody expressed pre-RXN post-RXN pre-RXN post-RXN pre-RXN post-RXND 3 × 10⁸ 2.7 × 10⁸ 3 × 10⁸ 3.0 × 10⁸ 3 × 10⁸ 3.0 × 10⁸ SV40 large 3 ×10⁸ 2.4 × 10⁷ 3 × 10⁸ 2.4 × 10⁸ 3 × 10⁸   2 × 10⁸ T antigen-gpD fusionprotein

The results indicated that most of the “LT-phage” had the NLS exposed onthe surface of the head, as evidenced by an approximately {fraction(1/10)} reduction in the titer (from 3×10⁸ to 2.4×10⁸).

Example 5 Nuclear Translocation Activities of the λ Phage Having NuclearLocalization Signals Exposed on the Head Surface

The plasmid which can express the fusion protein between SV40 large Tantigen and gpD was introduced into the E. coli TOP10 lysogenized by the80% genome D amber phage. The bacteria were cultured in the LB (10 mMMgSO₄, 100 μg/ml ampicillin) medium at 32° C., and at the logarithmicgrowth phase (2×10⁸ cells/ml) the culture was shaken at 45° C. for 20min to induce the phage production. Then the culture was shaken in thepresence of 1 mM IPTG for 3 hrs at 39° C., centrifuged at 5,000 rpm for10 min, and the precipitated E. coli cells were resuspended in theλ-buffer (10 mM Tris-HCl (pH 7.5), 10 mMMgSO₄, 0.01% gelatin, 10 mMputrescine). The concentrated bacteria were lysed at 37° C. by addingchloroform and stirring. Further, the solution was added with DNase, andcentrifuged at 8,000 rpm for 30 min to remove insoluble matters. Thesupernatant was added with 10% polyethylene glycol #6000 and 1 M NaCl,treated at 0° C. for 2 hrs, and centrifuged at 8,000 rpm for 30 min toprecipitate the phage. The phage was resuspended in the λ buffer and therecovered phage particles were purified by cesium chloridedensity-gradient ultracentrifugation.

The phage particles were dissolved in the λ-buffer to 2 mg/ml, andmicroinjection was carried out in the same manner as in Example 3 (3).The serum prepared by sensitizing the rabbit with the wild type λ phagetogether with Freund's adjuvant was used as the primary antibody fordetection. The results confirmed that while the wild type λ phage, usedas a control, did not exhibit the nuclear translocation activity (FIG.3, top), the λ phage with the nuclear localization signal hadaccumulated in the nucleus in 30 min (FIG. 3, bottom).

Industrial Applicability

The present invention provides the λ phage with a nuclear localizationsignal capable of packaging macromolecules such as long chain DNA andhaving a nuclear translocation activity. This phage can, for example,transport desired foreign genes, as long chain DNAs including theupstream regions, to the nucleus, and therefore it is expected to beutilized effectively in a variety of fields such as clarification ofbiological phenomena and gene therapy.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 12 <210> SEQ ID NO 1 <211> LENGTH: 20<212> TYPE: PRT <213> ORGANISM: Simian virus 40 <400> SEQUENCE: 1Lys Met Ala Pro Thr Lys Arg Lys Gly Ser Al #a Pro Gly Ala Ala Pro 1               5   #                10   #                15Lys Lys Pro Lys             20 <210> SEQ ID NO 2 <211> LENGTH: 33<212> TYPE: PRT <213> ORGANISM: Simian virus 40 <400> SEQUENCE: 2Tyr Asp Asp Glu Ala Thr Ala Asp Ser Gln Hi #s Ser Thr Pro Pro Lys 1               5   #                10   #                15Lys Lys Arg Lys Val Glu Asp Pro Lys Asp Ph #e Glu Ser Glu Leu Leu            20       #            25       #            30 Ser<210> SEQ ID NO 3 <211> LENGTH: 30 <212> TYPE: PRT<213> ORGANISM: Hepatitis D virus <400> SEQUENCE: 3Lys Lys Asp Lys Asp Gly Glu Gly Ala Pro Pr #o Ala Lys Lys Leu Arg 1               5   #                10   #                15Met Asp Gln Met Glu Ile Asp Ala Gly Pro Ar #g Lys Arg Pro            20       #            25       #            30<210> SEQ ID NO 4 <211> LENGTH: 7 <212> TYPE: PRT<213> ORGANISM: Simian virus 40 <400> SEQUENCE: 4Pro Lys Lys Lys Arg Lys Val   1               5 <210> SEQ ID NO 5<211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Bacteriophage lambda<400> SEQUENCE: 5 gtaagccatg gttatgacga gcaaag          #                   #              26 <210> SEQ ID NO 6 <211> LENGTH: 32<212> TYPE: DNA <213> ORGANISM: Bacteriophage lambda <400> SEQUENCE: 6gttcgaattc ctattaaacg atgctgattg cc        #                  #          32 <210> SEQ ID NO 7 <211> LENGTH: 22 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 7Met Lys Met Ala Pro Thr Lys Arg Lys Gly Se #r Ala Pro Gly Ala Ala 1               5   #                10   #                15Pro Lys Lys Pro Lys Thr             20 <210> SEQ ID NO 8<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM: Simian Virus 40<400> SEQUENCE: 8 Met Tyr Asp Asp Glu Ala Thr Ala Asp Ser Gl#n His Ser Thr Pro Pro  1               5   #                10  #                15 Lys Lys Lys Arg Lys Val Glu Asp Pro Lys As#p Phe Glu Ser Glu Leu             20       #            25      #            30 Leu Ser Thr         35 <210> SEQ ID NO 9<211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Simian Virus 40<400> SEQUENCE: 9 Met Lys Lys Asp Lys Asp Gly Glu Gly Ala Pr#o Pro Ala Lys Lys Leu  1               5   #                10  #                15 Arg Met Asp Gln Met Glu Ile Asp Ala Gly Pr#o Arg Lys Arg Pro Thr             20       #            25      #            30 <210> SEQ ID NO 10 <211> LENGTH: 18 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 10Met Ser Asp Arg Val Tyr Leu His Pro Phe Gl #y Ala Pro Ser Val Gly 1               5   #                10   #                15 Arg Thr<210> SEQ ID NO 11 <211> LENGTH: 16 <212> TYPE: PRT<213> ORGANISM: Simian Virus 40 <400> SEQUENCE: 11Met Pro Lys Lys Lys Arg Lys Val Gly Ala Pr #o Ser Val Gly Arg Thr 1               5   #                10   #                15<210> SEQ ID NO 12 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Simian Virus 40 <400> SEQUENCE: 12Met Pro Lys Lys Lys Arg Lys Val Thr  1               5

What is claimed is:
 1. A kit for transforming cells, comprising (a) abacterial host carrying a vector comprising a DNA encoding a fusionprotein comprising a nuclear localization signal and a λ phage headprotein, and (b) a λ phage that is incapable of expressing said λ phagehead protein in the bacterial host.
 2. The kit of claim 1, wherein saidλ phage head protein is the D protein of λ phage.